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Micah J. Guthrie, Jennifer Kang-Mieler; Development Of Double-barreled Nitric Oxide And Electroretinogram Microelectrode For Direct Measurement Of Nitric Oxide In The Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2469.
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Double-barreled microelectrodes have been developed to measure nitric oxide (NO) concentrations and the local electroretinogram (LERG) from the same location in the retina, with the LERG providing information about position in the retina.
Microelectrodes were constructed from double-barreled capillary glass pulled to an approximately 8 microns tip). One barrel contained a 5 micron diameter carbon fiber to detect NO and the other barrel is used to record LERGs and act as a counter/reference electrode to the carbon fiber. The carbon fiber is electrochemically etched to produce a conical tip approximately 15 microns in length. The etched tips are dip-coated with a Nafion® membrane to prevent interference from ascorbic acid and nitrite. The reference barrel is pressurized to 50 psi during the coating to prevent Nafion® from blocking the reference barrel. NO is amperometrically detected by a potentiostat with the carbon fiber held to 0.9 volts relative to a Ag/AgCl wire in the reference barrel. Electrodes were calibrated with various concentrations of S-nitroso-N-acetylpenicillamine, which spontaneously releases NO in aqueous solutions. The responses to different concentrations of ascorbic acid (AA) were used to determine the selectivity of the electrode for NO versus AA. LERGs were recorded from the reference/voltage barrel in an in-vivo rodent eyecup preparation to verify that barrel remained unblocked by Nafion®, with subdermal electrodes serving as reference and ground electrodes.
The average sensitivity of the Nafion® coated electrodes to was 2.29 ± 0.7 picoamps per µM NO, compared to 10.2 ± 1.3 picoamps per µM NO for uncoated electrodes. The selectivity of coated electrodes for NO versus AA ranged from 50 to 1000 with an average of 334 ± 243.8, compared to an average of 2.07 ± 0.02 for uncoated. Reference/voltage barrel remained unblocked after coating and able to record eyecup LERGs.
The developed electrodes are both selective for nitric oxide and capable of recording LERGs. The variability in selectivity is due to the nature of the Nafion® dipping process, and improving the uniformity of coating can increase the selectivity. The current data suggest that a double-barreled NO-LERG electrode can detect NO and measure LERGs and will be a beneficial tool in exploring the effects of NO on retinal function in in-vivo animal models.
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